Inhibition of Survival Pathways MAPK and NF-kB Triggers Apoptosis in Pancreatic Ductal Adenocarcinoma Cells via Suppression of Autophagy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a survival rate of 4-6 months from diagnosis. PDAC is the fourth leading cause of cancer-related death in the Western world, with a mortality rate of 10 cases per 100,000 population. Chemotherapy constitutes only a palliative strategy, with limited effects on life expectancy. AIMS: To investigate the biological response of PDAC to mitogen-activated protein kinase (MAPK) and NF-kappaB (NF-kB) inhibitors and the role of autophagy in the modulation of these signaling pathways in order to address the challenge of developing improved medical protocols for patients with PDAC. METHODS: Two ATCC cell lines, MIAPaCa-2 and PANC-1, were used as PDAC models. Cells were exposed to inhibitors of MAPK or NF-kB survival pathways alone or after autophagy inhibition. Several aspects were analyzed, as follows: cell proliferation, by [(3)H]TdR incorporation; cell death, by TUNEL assay, regulation of autophagy by LC3-II expression level and modulation of pro-and anti-apoptotic proteins by Western blot. RESULTS: We demonstrated that the inhibition of the MAPK and NF-kB survival pathways with U0126 and caffeic acid phenethyl ester (CAPE), respectively, produced strong inhibition of pancreatic tumor cell growth without inducing apoptotic death. Interestingly, U0126 and CAPE induced apoptosis after autophagy inhibition in a caspase-dependent manner in MIA PaCa-2 cells and in a caspase-independent manner in PANC-1 cells. CONCLUSIONS: Here we present evidence that allows us to consider a combined therapy regimen comprising an autophagy inhibitor and a MAPK or NF-kB pathway inhibitor as a possible treatment strategy for pancreatic cancer.

Synergism of arsenic trioxide and MG132 in Raji cells attained by targeting BNIP3, autophagy, and mitochondria with low doses of valproic acid and vincristine.

We previously demonstrated that arsenic trioxide (ATO) and proteasome inhibitor MG132 synergistically induced cell death in promonocytic leukaemia cell line U937 but were antagonistic in Burkitt's lymphoma cell line Raji. Here we explore the role of autophagy, expression of BNIP3, and mitochondrial mass, in determining whether ATO and MG132 interaction can be shifted from antagonism to synergism in Raji cells. Treatment with ATO+MG132 increased the percentage of cells with collapsed mitochondrial membrane potential (MMP) in U937 cells, but had no effect in Raji cells. Mitochondria were found in cytoplasmic marginal location in U937 cells but at perinuclear location in Raji cells. ATO+MG132 increased mitochondrial mass in U937 cells but decreased it in Raji cells, while autophagy was increased in both cell lines. BNIP3 was expressed in U937 cells at cytoplasmic marginal locations and was hardly detected in Raji cells. Histone deacetylase (HDAC) inhibitor valproic acid (VPA) increased expression of BNIP3 in Raji cells at perinuclear locations. However antagonism between ATO and MG132 was increased in the presence of low doses of VPA. Addition of vincristine (VCR) blocked autophagy, while VPA+VCR treatment of Raji cells at sub-cytotoxic doses caused BNIP3 and mitochondria to redistribute to cytoplasmic peripheral location and increased mitochondrial mass. ATO+MG132 in the presence of subcytotoxic doses of VPA+VCR caused collapse of MMP in Raji cells, while interaction between ATO and MG132 shifted from antagonism to synergism. We conclude that synergism between ATO and MG132 was attained in Raji cells by disruption of the perinuclear mitochondrial cluster, blockage of selective autophagy of mitochondria (mitophagy) by VCR, increased mitochondrial mass, and upregulation of BNIP3 by VPA.

The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav.) Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm) and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion.

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O2⁻) levels. Our results showed that combined arsenite+MG132 produced low levels of O2⁻ at 6h and 24h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80µM hydrogen peroxide together with arsenite+MG132 changed synergism on cell death to antagonism at all effect levels while increasing O2⁻ levels. Arsenite+hydrogen peroxide also resulted in antagonism with increased O2⁻ levels in U937 cells. In Raji cells, arsenite+MG132 also produced low levels of O2⁻ at 6h and 24h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite+CAPE showed high levels of O2⁻ production at 6h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O2⁻ levels at early time points after exposure.

Trichinella infection in wild boars (Sus scrofa) from a protected area of Argentina and its relationship with the presence of humans.

In Argentina, Trichinella infection has been documented in humans and animals of several provinces since 1930. This zoonotic parasite infection has been recently detected in humans and pigs of a region historically considered as Trichinella-free, suggesting the spread of these pathogens. The aim of the present work was to investigate the presence of Trichinella infection in wild boars (Sus scrofa) and in the human population living in a protected area. Trichinella infection has been investigated by serology (in humans and wild boars) and by artificial digestion of wild boar muscles. The isolated Trichinella larvae have been identified at the species level by multiplex PCR. A geographical information system has been used to collect environmental data. The results showed the circulation of Trichinella spiralis in wild boars with a low parasite burden, and suggest the influence of human behavior on the transmission. The transplacental passage of parasite is postulated. It follows that the declaration of region as Trichinella-free should be carefully established by means of extensive monitoring programs, not only in humans and domestic animals but also in wildlife.

[Trichinellosis serology in blood-donors from a non-endemic area of Argentina]. / Serología para triquinosis en dadores de sangre en área no endémica de la Argentina.

The aim of the present study was to determine the presence of human trichinellosis in an area of Argentina historically regarded as free of this parasitic disease. This parasitosis was assessed on 150 blood donors by means of immunoserological tests, and their relation with risk alimentary habits by a questionnaire. The questionnaires showed that 98.0% (n=147) of the individuals consumed pork and pork products at a high frequency. The main pork products incorporated to the habitual diet were the stuffed ones. A 80.3% (n=118) of the individuals had acquired such products at butchers, a 38.1% (n=56) at home slaughters and a 34.0% (n=50) at supermarkets. The immunoserological techniques employed were: enzyme immunoanalysis, indirect immunofluorescence and/or immunoelectrotransfer blot assay. Anti-Trichinella antibodies were detected in 8.0% (n=12) of the serum samples by more than one of the methodologies employed, considering these results as confirmatory of trichinellosis. The studies carried out herein demonstrate the existence of human trichinellosis in an area historically regarded as free of this parasitic disease and suggest its relationship with the ingestion of pork or pork products as a risk factor. These results indicate that the lack of reports should not be the only criterion for an area to be considered as Trichinella-free. The awareness of the existence of the parasite in a region will favor the establishment of control and prevention strategies which is of fundamental importance to avoid the spread of the disease.

Serología para triquinosis en dadores de sangre en área no endémica de la Argentina / Trichinellosis serology in blood-donors from a non-endemic area of Argentina

El presente estudio tuvo como objetivo determinar la presencia de triquinosis humana en un área considerada históricamente libre de esta parasitosis en la Argentina. Se evaluó la parasitosis mediante técnicas inmunoserológicas y su relación con los hábitos alimenticios de riesgo mediante un cuestionario realizado a 150 donantes de sangre. Las encuestas revelaron que el 98.0% (n=147) de los individuos consumía carne de cerdo y/o derivados con elevada frecuencia. Los principales productos porcinos incorporados a la alimentación habitual fueron los chacinados. El 80.3% (n=118) de los individuos adquirían los productos en carnicerías, el 38.1% (n=56) en faenas domiciliarias y el 34.0% (n=50) en supermercados. Las técnicas inmunoserológicas utilizadas para el diagnóstico de triquinosis fueron: enzimoinmunoanálisis, inmunofluorescencia indirecta y/o inmunoelectrotransferencia. En el 8.0% (n=12) de los sueros de los individuos se detectaron anticuerpos anti-Trichinella por más de una de las técnicas realizadas, considerándose este resultado diagnóstico confirmatorio de triquinosis. Los estudios realizados demuestran la existencia de triquinosis humana en un área considerada libre de esta parasitosis y sugieren su relación con la ingesta de carne porcina y/o sus subproductos, hábito alimenticio de riesgo. Estos resultados indican que la falta de denuncia de casos de la enfermedad no debe ser el único criterio a tener en cuenta para considerar a una región área libre de triquinosis. El conocimiento de la presencia del parásito en un área determinada favorece la instauración de medidas de control y prevención evitando la diseminación de la parasitosis.

The aim of the present study was to determine the presence of human trichinellosis in an area of Argentina historically regarded as free of this parasitic disease. This parasitosis was assessed on 150 blood donors by means of immunoserological tests, and their relation with risk alimentary habits by a questionnaire. The questionnaires showed that 98.0% (n=147) of the individuals consumed pork and pork products at a high frequency. The main pork products incorporated to the habitual diet were the stuffed ones. A 80.3% (n=118) of the individuals had acquired such products at butchers, a 38.1% (n=56) at home slaughters and a 34.0% (n=50) at supermarkets. The immunoserological techniques employed were: enzyme immunoanalysis, indirect immunofluorescence and/or immunoelectrotransfer blot assay. Anti-Trichinella antibodies were detected in 8.0% (n=12) of the serum samples by more than one of the methodologies employed, considering these results as confirmatory of trichinellosis. The studies carried out herein demonstrate the existence of human trichinellosis in an area historically regarded as free of this parasitic disease and suggest its relationship with the ingestion of pork or pork products as a risk factor. These results indicate that the lack of reports should not be the only criterion for an area to be considered as Trichinella-free. The awareness of the existence of the parasite in a region will favor the establishment of control and prevention strategies which is of fundamental importance to avoid the spread of the disease.

Detection of trichinellosis in a historically Trichinella-free area of Argentina.

The aim of the present work was to determine the presence of human and porcine trichinellosis in an area of Argentina historically regarded as Trichinella-free. Human blood donors (n=216) and swine destined for consumption (n=57) were evaluated by serological techniques (ELISA, immunofluorescence, and/or Western Blot). Muscle tissues from 26 of the pigs were evaluated for the presence of Trichinella larvae by the artificial digestion method. A questionnaire was used to collect and evaluate data on eating habits of the human population under study and on swine-raising conditions. The survey showed that 98.1% of the individuals (n=212) were regular consumers of pork in the form of stuffed products such as sausages produced by local butchers. The seroprevalence (positive sera by at least two of the three methods) was 8.3% (n=18) for human trichinellosis and 24.5% (n=14) for porcine trichinellosis. Trichinellaspiralis larvae were found in 2 of the 26 pigs (7.7%) with parasite loads of 0.33 and 2.4 muscle larvae per gram. Twelve swine found positive by serological and/or parasitological tests were raised under poor sanitary conditions (presence of rubbish in the surroundings, with cannibalism and scavenging behaviors, presence of rodents, etc.). Our study confirms the existence of porcine trichinellosis in an area regarded as Trichinella-free, provides supporting serological evidence of human infection in this area, and indicates that failure to report cases of trichinellosis based on inadequate surveillance can result in incorrect prevalence classification of an area.

Evaluation of three immunoserological techniques in the detection of porcine trichinellosis.

Three immunoserological tests (IST) used for the detection of porcine trichinellosis, immunofluorescence (IF), enzyme-inmunoanalysis (EIA), and Western blot (WB), were compared. Three groups of animals were analyzed: Group 1, animals naturally infected with parasite burdens (PB) of <1 muscle larvae (ML)/g (n=18); Group 2, animals naturally infected with PB of > or =2ML/g (n=23); Group 3, animals raised and home-slaughtered on farms in Argentina (n=59). Animals from Groups 1 and 2 were identified in outbreaks and were analyzed by individual artificial digestion (AD) of > or =30g of muscle. Animals in Group 3 were subjected to AD of 5g of muscle. The detection percentages in sera of swine with the lower PB were 100% for IF, 72% for EIA, and 50% for WB. Eighty-three percent of the animals were serologically positive by two or three techniques. In pigs with the higher PB, the detection percentage was similar for IF and EIA (100% vs. 91%, respectively), and was lower for the WB (61%). Ninety-six percent of the animals were serologically positive by two or three techniques. Group 3 animals had similar detection percentages for the three techniques (IF, 30%; EIA, 29%; WB, 42%). Twenty-five percent of the animals were serologically positive by two or three techniques. Two animals were positive by AD with PB of 0.33 and 2.4ML/g, and were positive for IF and WB, or IF, EIA, and WB. Results indicate that the sensitivity of each technique depends on the PB, and always ranked in sensitivity as IF>EIA>WB. For the lower PB, the decrease in the sensitivity is more pronounced for the EIA. Although the WB has a low sensitivity, the detection of the specific bands for Trichinella spiralis makes it a useful confirmatory tool. Considering that more than 83% of the parasitologically positive animals had 2 or 3 positive serological results using the techniques tested here, for the diagnosis of porcine trichinellosis, pigs positive by two of these serological techniques must be regarded as truly infected pigs.

Immunoparasitological evaluation of Trichinella spiralis infection during human pregnancy: a small case series.

The clinical background, vertical transmission of Trichinella spiralis and helminthotoxic activity of sera were evaluated in four pregnant women infected at different gestation times. Parasitological and immunological parameters were analyzed. Patients presented clinical symptoms and a seroconversion pattern typical of Trichinella infection, independently of the gestation trimester. All patients, including one patient treated with mebendazole, gave birth to healthy infants at full term. Studies performed in placentas and umbilical cords by artificial digestion and/or immunofluorescence did not reveal the presence of parasites or parasite antigens. Specific antibodies were found in placental extraction and maternal sera by immunofluorescence, ELISA and/or immunoelectrotransfer blot assay. Specific IgG, IgE and IgA were found in the umbilical cord sera. One umbilical cord demonstrated immune-complexed specific IgM and circulating parasite antigens were found in the corresponding infant up to 10 months after birth. Patients' sera were able to kill newborn larvae in cytotoxicity assays, even in the absence of specific antibodies; this effect was abrogated by mifepristone. The results suggest that in human trichinellosis during pregnancy there is an enhanced helminthotoxicity to newborn larvae, dependent in part on progesterone, leading to a mild or moderate course of the infection. The results also indicate that the transplacental passage of migrating larvae is possible.

[Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina. The acute and chronic phases of the infection]. / Aspectos clínicos, serológicos y parasitológicos de un brote de triquinelosis humana en Villa Mercedes, San Luis, Argentina. Las fases aguda y crónica de la infección.

Clinical and serological features of individuals undergoing the acute (n: 54) and chronic (n: 32) phases of trichinellosis belonging to an outbreak originated by the consumption of pork products of a commercial source, arisen in Argentina, are described. Epidemiological data, signs, symptoms, and laboratory studies were assessed. Parasitological studies were performed in patients and pork products. Results showed that: a) the parasite burden of pork products was 200.0 +/- 18.3 larvae/g; b) muscle larvae were demonstrated in 10 out of the 11 patients studied; c) during the acute phase, fever (94%), eosinophilia (90%), myalgia (85%), headache (81%), facial edema (54%), diarrhoea (52%) and anti-Trichinella antibodies (64%) were observed; d) 15% of the patients had to be hospitalized, 7% having complications; e) during the chronic phase, myalgia (72%), sight abnormalities (22%), gastrointestinal disorders (31%) and the persistence of antibodies (77%) were observed. This study highlights the socioeconomical impact of trichinellosis due to the clinical characteristics of the acute phase and the presence of symptoms in the chronic phase.

Aspectos clínicos, serológicos y parasitológicos de un brote de triquinelosis humana en Villa Mercedes, San Luis, Argentina. Las fases aguda y crónica de la infección. / [Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina. The acute and chronic phases of the infection]

Clinical and serological features of individuals undergoing the acute (n: 54) and chronic (n: 32) phases of trichinellosis belonging to an outbreak originated by the consumption of pork products of a commercial source, arisen in Argentina, are described. Epidemiological data, signs, symptoms, and laboratory studies were assessed. Parasitological studies were performed in patients and pork products. Results showed that: a) the parasite burden of pork products was 200.0 +/- 18.3 larvae/g; b) muscle larvae were demonstrated in 10 out of the 11 patients studied; c) during the acute phase, fever (94

), eosinophilia (90

), myalgia (85

), headache (81

), facial edema (54

), diarrhoea (52

) and anti-Trichinella antibodies (64

) were observed; d) 15

of the patients had to be hospitalized, 7

having complications; e) during the chronic phase, myalgia (72

), sight abnormalities (22

), gastrointestinal disorders (31

) and the persistence of antibodies (77

) were observed. This study highlights the socioeconomical impact of trichinellosis due to the clinical characteristics of the acute phase and the presence of symptoms in the chronic phase.

Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires / Serological, clinical and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires

Se estudió un brote de triquinosis ocurrido en el sur de la provincia de Buenos Aires, zona endémica para esta zoonosis. Dieciocho personas fueron afectadas por la ingesta de productos elaborados con carne de cerdo (chacinados sin el correspondiente control veterinario. Se describen síntomas y signos de los apcientes, como así también estudios serológicos relacionados con la formación de anticuerpos y la capacidad citotóxica de los sueros. Se analizaron los producto elaborados (chacinados), así como ocho cerdos interdictos de la granja donde se originó el brote. Con el fin de estudiar la cadena de transmisión se capturaron y analizaron ratas de los alrededores del hábitar de los porcinos. Este trabajo demuestra que los principales síntomas y signos fueron: mialgia, edema palpebral, fiebre y cefalea, que el 61// de los pacientes presentó anticuerpos contra el estadio de larva muscular entre los 15-30 días posteriores a la ingesta, que sólo presentaron anticuerpos contra la larva recien nacida a los 40-60 días postinfección cinco de los dieciocho pacientes, que la carga parasitaria en los chacinados fue de 5,3 larvas/g, que dos de los ocho cerdos se diagnosticaron por técnicas serológicas, mientras que por técnicas parasitológicas se detectó sólo uno y que la rata es uno de los principales reservorios en la cadena epidemiológica

Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires / Serological, clinical and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires

Se estudió un brote de triquinosis ocurrido en el sur de la provincia de Buenos Aires, zona endémica para esta zoonosis. Dieciocho personas fueron afectadas por la ingesta de productos elaborados con carne de cerdo (chacinados sin el correspondiente control veterinario. Se describen síntomas y signos de los apcientes, como así también estudios serológicos relacionados con la formación de anticuerpos y la capacidad citotóxica de los sueros. Se analizaron los producto elaborados (chacinados), así como ocho cerdos interdictos de la granja donde se originó el brote. Con el fin de estudiar la cadena de transmisión se capturaron y analizaron ratas de los alrededores del hábitar de los porcinos. Este trabajo demuestra que los principales síntomas y signos fueron: mialgia, edema palpebral, fiebre y cefalea, que el 61// de los pacientes presentó anticuerpos contra el estadio de larva muscular entre los 15-30 días posteriores a la ingesta, que sólo presentaron anticuerpos contra la larva recien nacida a los 40-60 días postinfección cinco de los dieciocho pacientes, que la carga parasitaria en los chacinados fue de 5,3 larvas/g, que dos de los ocho cerdos se diagnosticaron por técnicas serológicas, mientras que por técnicas parasitológicas se detectó sólo uno y que la rata es uno de los principales reservorios en la cadena epidemiológica (AU)

Estudio sobre las condiciones de fijación de IgG a la proteína A de Staphylococcus aureus / The conditions of IGG fixation to the protein A of Staphylococcus aureus

Distintas cepas de Staphylococcus aureus tienen una proteína de membrana denominada "proteína A", la que fija IgG1, IgG2, e IgG4 humanas y la IgG de otras especies animales. La unión tiene lugar mediante el fragmento Fc. Debido a esta propiedad, el Staphylococcus aureus, que sintetiza proteína A, ha sido empleado para la adsorción específica de anticuerpos contra diferentes bacterias y utilizados para el diagnóstico microbiológico. Teniendo en cuenta las ventajas de este método, se estudian en este trabajo las condiciones físico-químicas que regulan la fijación de IgG a la proteína A de 33 cepas de Staphylococcus aureus coagulasa positivas. De acuerdo con estos resultados se propone un método adecuado para la unión de la IgG a la proteína A bacteriana

Estudio sobre las condiciones de fijación de IgG a la proteína A de Staphylococcus aureus / The conditions of IGG fixation to the protein A of Staphylococcus aureus

Distintas cepas de Staphylococcus aureus tienen una proteína de membrana denominada "proteína A", la que fija IgG1, IgG2, e IgG4 humanas y la IgG de otras especies animales. La unión tiene lugar mediante el fragmento Fc. Debido a esta propiedad, el Staphylococcus aureus, que sintetiza proteína A, ha sido empleado para la adsorción específica de anticuerpos contra diferentes bacterias y utilizados para el diagnóstico microbiológico. Teniendo en cuenta las ventajas de este método, se estudian en este trabajo las condiciones físico-químicas que regulan la fijación de IgG a la proteína A de 33 cepas de Staphylococcus aureus coagulasa positivas. De acuerdo con estos resultados se propone un método adecuado para la unión de la IgG a la proteína A bacteriana (AU)